Evolution of Viola stagnina and its sisterspecies by hybridisation and polyploidisation Hof, K. van den

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Evolution of Viola stagnina and its sisterspecies by hybridisation and polyploidisation

Hof, K. van den

Citation

Hof, K. van den. (2010, June 9). Evolution of Viola stagnina and its sisterspecies by hybridisation and polyploidisation. Retrieved from https://hdl.handle.net/1887/15684

Version: Corrected Publisher’s Version

License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden

Downloaded from: https://hdl.handle.net/1887/15684

Note: To cite this publication please use the final published version (if applicable).

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Evolution of Viola stagnina and its Sisterspecies by

Hybridization and Polyploidization

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Violen

... ze zijn dapper; fragiel aan alle kanten, maar toch staan ze op wacht. Ze bewaken als het ware het voorjaars-

gevoel. Ze symboliseren nederigheid en trouw.

Kevin van den Hof

Evolution of Viola stagnina and its Sisterspecies by Hybridization and Polyploi- dization

Cover design and

lay-out : René Glas (www.reneglas.com) Drawings, fig. 9 : Esmée Winkel

Photograph, fig.17 : Bertie-Joan van Heuven Printed by : Offsetdrukkerij Nautilus, Leiden

Chapter 2 : Published in Molecular Biology and Evolution 25:

2099–2108 (2008). van den Hof K, van den Berg RG, Gravendeel B. Chalcone synthase gene lineage diversifica tion confirms allopolyploid evolutionary relationships of European rostrate violets.

Chapter 3 : Submitted to Taxon Chapter 4 : Submitted to Taxon

Chapter 5 : Submitted to Conservation Genetics Chapter 6 : Submitted to Plant Ecology and Evolution

No part of this publication, apart from bibliographic data and brief annotations in critical reviews, may be reproduced, re-recorded or published in any form, including print, photocopy, microform, electronic or electromagnetic record without written permission by the publishers.

ISBN/EAN: 978-90-71236-71-6

Dedicated to the memory of

Ruud van der Meijden

Martin Bril - Volkskrant - 5 april 2006

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Evolution of Viola stagnina and its Sisterspecies by Hybridization and Polyploidization

Proefschrift

ter verkrijging van de graad van Doctor aan de Universiteit Leiden,

op gezag van de Rector Magnificus prof. mr. P.F. van der Heijden, volgens het besluit van het College van Promoties

te verdedigen op woensdag 9 juni 2010 klokke 16:15 uur

door

Kevin van den Hof

Geboren te Geleen in 1980

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P r o m o t i e c o m m i s s i e

Promotor • Prof. Dr. E.F. Smets

Copromotor • Dr. B. Gravendeel Dr. R.G. van den Berg

Referent • Prof. Dr. H.E. Ballard Jr.

Overige leden • Prof. Dr. P. Baas Dr. T. Marcussen Dr. M.E. Noordeloos Prof. Dr. M. Schilthuizen

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C o n t e n t s

Chapter 1 • General introduction 7

Chapter 2 • Chalcone Synthase Gene Lineage Diversification confirms Al- lopolyploid Evolutionary Relationships of European Rostrate Violets

13

Chapter 3 • Viola montana and V. persicifolia (Violaceae): two names to be

rejected 27

Chapter 4 • Proposal to reject the names Viola montana and V. persicifolia

(Violaceae) 41

Chapter 5 • Combined analyses of AFLP markers and morphology confirm the taxonomic status of Viola stagnina var. lacteoides 45 Chapter 6 • Phenotypic plasticity of Viola stagnina 65

Chapter 7 • Summary & Conclusion 75

Nederlandse samenvatting 79

References 83

Appendix I • Voucher information of taxa sampled 95

Appendix II • List of species and accessions used for AFLP analyses 99

Appendix III• List of characters used for morphometric analyses 105

Curriculum Vitae 109

Nawoord 111

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7

Chapter

1

General introduction

I

n this thesis the patterns resulting from hybridization and polyploidization in a group of closely related Viola species were investigated. The status of the two infraspecific taxa within V. stagnina was studied in detail, and the nomenclature of a number of taxa was investigated.

Species concept

A never ending discussion in the field of biology is that of the species concept.

Numerous papers and books have dealt with this subject, but no consensus about this definition exists among biologists. The fact that so many have discussed this subject is probably because a species is considered to be the most fundamental unit of comparison in all fields of biology and it is therefore the most important term used (de Queiroz, 2005).

Before the publication of Charles Darwin’s book on the origin of species (1859), taxonomists had discussions about what a species defines. In those days, there was a more or less essentialist view on what a species was. Species were considered to be fixed entities that could not change over time. Discussions on species definition were in fact taxonomic puzzles about whether one was dealing with a species or a variety (Hey, 2006).

This can be illustrated with an example in Viola. Some 19^th century botanists treated V.

lactea, V. pumila and V. stagnina as variations within a single species (e.g. Reichenbach, 1823), while others treated them as three separate species (e.g. Koch, 1836). A discussion about the species concept itself however did not exist.

After Charles Darwin presented his theory of evolution, the definition of a species became more prevalent and complicated, because the theory made biologists realize that all living organisms are subjected to evolution by means of adaptation and natural selection.

This meant that varieties could now change into species over time. The boundary between a variety and a species became therefore not only vague, as it was for the 19^th century essentialists, but it also became dynamic. Classifying species now became subjective and arbitrary (Hey, 2001; 2006).

The species concept had become a dilemma. It is in human’s nature to classify the surrounding world, in the case of biologists this means classifying organisms. But biologists have a problem because the items they are classifying are changing over time, they are evolving. Biologists therefore started treating species as a “group of organisms enjoined by evolutionary processes that go on within it, and that is separate from other groups because of the absence of shared evolutionary processes with those other groups” (Hey, 2001). The focus on evolutionary processes within has led to the development of at least two dozen species concepts where species are defined by referring to evolutionary processes (e.g.

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Cracraft, 1983, Cronquist, 1988; Kornet and McAllister, 1993; Mayden, 1997; Mayr, 1969;

Templeton 1989; Van Valen, 1976; Wiley, 1978). These species concepts, however, are not applicable to all living organisms since it is seemingly impossible to incorporate the multitude of evolutionary processes driving speciation in one comprehensive definition.

These evolutionary processes are just different ways used to describe what a species is, which shows that the species concept essentially is a human construct. It is therefore very unlikely that there ever will be a comprehensive definition for a species (Dobzhansky, 1955).

Still, biologists use species every day. They have to, because species are the most fundamental units of comparison in biology. It is therefore necessary to keep in mind that the concept one chooses to use is just a practical hypothesis. The working hypothesis used in this thesis is based on the phylogenetic species concept (Cracraft, 1983; Nixon and Wheeler, 1990). This concept defines species as the smallest aggregation of populations (sexual) or lineages (asexual) diagnosable by a unique combination of character states in comparable individuals. A character state is an inherited attribute distributed among all comparable individuals of the same historical population, clade, or terminal lineage (monophyletic group).

In this thesis, we also encountered variation below the species level. Definitions of infraspecific ranks are an even bigger hornets’ nest of contradicting opinions and concepts than that of the species concept itself (McDade, 1995; Stuessy, 1990) and most practical systematists and taxonomists try to avoid these ranks whenever possible. In some cases, however, complex patterns observed within a species demand using infraspecific ranks.

In this thesis, two different infraspecific ranks are recognized below the species level:

i.e. subspecies and variety. Subspecies differ from each other by at least one diagnosable character and are geographically separated from each other. The same definition is used for a variety, except that varieties are not geographically separated from each other (Stuessy, 1990). By recognizing infraspecific taxa, we acknowledge the existence of deviating populations. We feel that these populations deserve attention because they might eventually evolve into new species. Because we cannot witness this process within a human lifetime, this does not mean we should not recognize and describe them already.

In our view, though, the recognition of infraspecific taxa should be based on analyses of both molecular data and morphology in combination with common garden experiments.

Speciation by hybridization and polyploidization

Interspecific hybridization is seen as a common process and important mechanism for speciation in flowering plants (Grant, 1981; Ellstrand and Schierenbeck, 2000; Hegarty and Hiscock, 2004). Two forms of hybrid speciation are commonly recognized: homoploid speciation and alloploid speciation. Homoploid speciation involves the hybridization between two closely related taxa without a change in ploidy, resulting in more or less fertile offspring (Rieseberg, 1997; Rieseberg et al., 2003; Abbott et al., 2005). Alloploid speciation on the other hand usually involves hybridization between more distantly related taxa, which produces sterile offspring. The hybrid offspring then regains its fertility by doubling its chromosomes, which is called allopolyploidy. The resulting polyploid hybrid can have two or more sets of chromosomes derived from different parental species (Stebbins, 1971; Song et al., 1995; Bennett, 2004; Hegarty and Hiscock, 2004).

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9 As a consequence of hybridization, allopolyploidy, but also autopolyploidy (doubling of chromosomes without hybridization) have been important factors in the evolutionary history of plants (Grant, 1981; Soltis and Soltis, 2000). Almost all flowering plants and ferns have experienced at least one polyploidization event in their evolutionary history (Soltis et al., 2009). It is estimated that approximately 15% of the speciation events in flowering plants and 31% of the speciation events in ferns are accompanied by an increase in ploidy (Wood et al., 2009).

The study species: Viola stagnina and relatives

The violet family (Violaceae) consists of about 900 species divided in ca. 22 genera (Tokuoka, 2008). Viola is the largest genus with approximately 500 species. In contrast to most other genera of the Violaceae, which have a subtropical and tropical distribution, Viola species mainly have a northern temperate distribution (Ballard et al., 1999). The primary centers of taxonomic and morphological diversity can be found in the Alps and the Mediterranean, the Himalayas, montane eastern Asia, Patagonia and the South American Andes from where the genus is believed to have originated (Clausen, 1929; Valentine, 1962; Ballard et al., 1999).

Viola species are usually herbaceous plants with zygomorphic flowers. The flowers that fully open i.e. chasmogamous flowers possess adaptations to a wide range of temperate pollinators such as solitary bees, bumblebees, bombyliids and butterflies (Beattie, 1974). Next to these insect pollinated flowers, many species also produce self- pollinating (cleistogamous) flowers later in the season, as an extra reproductive assurance when insects are scarce (Redbo-Torstensson and Berg, 1995). Having developed this reproductive strategy during evolution is probably one of the key aspects responsible for the successful distribution of Viola (Clausen, 1929; Valentine, 1962).

Two other key aspects explaining the evolutionary success of Viola are hybridization and polyploid evolution and numerous reports have described such events in Viola (e.g.

Valentine, 1958; Moore and Harvey, 1961; Harvey, 1966; Ballard, 1993; Røren et al, 1994; Erben, 1996; Neuffer et al., 1999; Jonsell et al., 2000; Marcussen and Borgen, 2000;

Marcussen et al., 2001; Marcussen et al., 2005). In fact, the first report of an infrageneric series of polyploid levels was from Viola (Miyaji, 1913).

Viola stagnina (Fen violet) is a widespread but rare plant species occurring throughout Europe with the exception of the Mediterranean, the southeast and north of Europe (Fig. 10).

It favours wet and temporarily flooded, sunny habitats such as floodplains, fens and marshes.

(Valentine et al., 1968; Eckstein et al., 2006a; Weeda, 2002). Within Viola, V. stagnina is placed in sect. Viola subsect. Rostratae Kupffer (also known as section Trigonocarpea Godr.). This subsection consists of approximately 50 species with a northern temperate distribution in North America and Eurasia. Subsection Rostratae is characterized primarily by primitive characters. Previous phylogenetic studies using nrITS sequences have shown that the subsection is paraphyletic with respect to a number of other north-temperate groups (Ballard et al. 1999; Yoo et al. 2005). In Europe, where subsection Rostratae is morphologically most diverse, the subsection has traditionally been subdivided into four morphologically defined groups, here referred to as series. These series are the Arosulatae, Mirabiles, Repentes, and Rosulantes.

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Viola stagnina is placed in the Arosulatae series. This series consists of a group of five western Eurasian species. Species of the series are adapted to temporarily flooded habitats, rather than woodland, and are easily characterized by their leaf and stipule characters and the lack of a leaf rosette. The basal chromosome number of subsection Rostratae is x = 5, and since no diploid species (2n = 10) are known for this subsection, V. stagnina is considered to be a paleotetraploid with 2n=20 chromosomes (Marcussen and Nordal, 1998). Viola canina, V. elatior, and V. pumila are octoploids with 2n=40 and V. lactea is a subdodecaploid with 2n=58 chromosomes (Moore and Harvey, 1961). Cytological studies have shown that V. stagnina is involved as one of the parental species in the autoploid and alloploid origin of the other arosulate Violets (Fig. 1). Viola canina, V. pumila, and V. lactea are all alloploids, which have V. stagnina as one of the parental contributors to their alloploid genome (Moore and Harvey, 1961), while V. elatior is considered to be an autoploid derivative of V. stagnina (Clausen, 1927). The other parental species contributing to the alloploid genomes of the arosulate violets are likely to be extinct.

The varieties within V. stagnina

In the Netherlands, two morphs of V. stagnina have been described: V. stagnina var. stagnina and V. stagnina var. lacteoides W. Becker and Kloos (1924). The second variety was mentioned for the first time by Kloos (1924). He reported finding specimens resembling V. stagnina but being smaller in habit and having darker colored and thicker leaves. After having consulted Becker he concluded that he had found a new morph which he named V. persicifolia var. lacteaeoides. Dutch botanists after Kloos, however, had different opinions about the subdivision of V. stagnina into two infraspecific taxa, and after appearing in the flora of Heimans et al. (1924) and in the Heukels’ Schoolflora voor Nederland (1927) the variety disappeared from subsequent Dutch floras until 1977.

The varieties were mentioned again in the Heukels’ flora (van Oostroom, 1977), this time as subspecies. Den Held described subsp. lacteoides in the addenda and added that its stigma is straight as compared to hooked in V. stagnina subsp. stagnina, and that the spur of subsp. lacteoides exceeds the appendices on the calyx, whereas the spur of V. stagnina subsp. stagnina normally does not exceed these. The next edition of the Heukels’ flora (van der Meijden, 1983) noted that the taxonomy of the species was being investigated and that the infraspecific taxa within V. stagnina were being treated as varieties again until further notice. In the next edition of the Heukels’ flora (1990) no infraspecific taxa were recognized for V. stagnina anymore because Van der Meijden considered the differences between the morphs too small. Weeda (2001, 2002) devoted two papers to V.

stagnina in the Netherlands. Strongly disagreeing with van der Meijden (1990), Weeda pleaded for a resurrection of the subdivision of V. stagnina into two varieties based on the morphological differences mentioned by Kloos (1924) and den Held (in van Oostroom, 1977), but also because in the Netherlands both morphs of V. stagnina have a different geographical distribution with only a small overlap. The common stagnina morph is found in the Holocene part of The Netherlands where it mainly grows in fen meadows and on the floodplains of river and brook valleys. The main distribution of the lacteoides morph, on the other hand, is restricted to the Pleistocene part of the Netherlands. There it is mainly found in the valley of the IJssel river on the lower parts of wet heath lands on loamy and peaty soil (Weeda, 2001). Since the lacteoides morph has not been found outside The Netherlands, this is probably the first endemic plant for the Netherlands. Investigating its

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11 taxonomic status with molecular biological techniques is therefore not only interesting from a scientific point of view, but also important for conservation management, since the lacteoides morph has a very limited distribution area and needs active conservation management for its preservation.

Research questions

In this thesis, infraspecific variation within V. stagnina and hybridization and polyploidization between V. stagnina and its closest relatives were investigated to answer the following research questions:

Which species are most closely related to V. stagnina?

Can reticulate patterns of evolution between V. stagnina and its closest relatives be determined by using the low copy nuclear Chalcone Synthase (CHS) marker?

How many duplication events of CHS have taken place during the evolution of Viola?

Are V. persicifolia and V. montana the appropriate scientific names to use?

Is V. stagnina var. lacteoides genetically distinct from the more common V. stagnina var. stagnina?

Are there morphological traits separating the two morphs of V. stagnina from each other?

Thesis Goal & Outline

In chapter 2, the results of a phylogenetic study are presented in which the closest relatives of V. stagnina are determined including their reticulate relationships by using sequences of the CHS gene. This study also presents the evolutionary history of the CHS gene itself within the angiosperms.

In chapter 3, the nomenclatural history of the scientific names V. persicifolia Schreb.

(1771) and V. montana L. (1753) are discussed. In order to give priority to the names V.

stagnina and V. elatior, we propose to reject the older name V. persicifolia and V. montana respectively in chapter 4.

In chapter 5, we aim to determine the taxonomic status of the lacteoides morph of V. stagnina by studying the morphological and genetic variation of different populations. In chapter 6, a common garden experiment, a crossing experiment and a chromosome count of both varieties are described. Also the nomenclature of the lacteoides morph for both the scientific and vernacular epithets is discussed.

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2.

3.

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13

Chapter

2

Chalcone Synthase Gene Lineage Diversification

confirms Allopolyploid Evolutionary Relationships of European Rostrate Violets

¹

P

hylogenetic relationships among and within the subsections of the genus Viola are still far from resolved. We present the first organismal phylogeny of predominantly western European species of subsection Rostratae based on the plastid trnS-trnG intron and intergenic spacer and the nuclear low-copy gene Chalcone Synthase (CHS) sequences. CHS is a key enzyme in the synthesis of flavonoids, which are important for flower pigmentation. Genes encoding for CHS are members of a multigene family. In Viola, three different CHS copies are present. CHS gene lineages obtained confirmed earlier hypotheses about reticulate relationships between species of Viola subsection Rostratae based on karyotype data. Comparison of the CHS gene lineage tree and the plastid species phylogeny of Viola reconstructed in this study indicates that the different CHS copies present in Viola are the products of both recent and more ancient duplications.

Key words: Chalcone synthase, gene lineage diversification, phylogeny, Viola subsection Rostratae, allopolyploidy, trnS-trnG.

K. van den Hof, R.G. van den Berg and B. Gravendeel

1Published as: van den Hof et al., 2008. Mol. Biol. Evol. 25: 2099-2109.

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Introduction

Speciation through hybridization is considered a common process in higher plants.

Although hybrids between distantly related taxa are usually sterile, they can become fertile again by doubling their chromosome numbers. The resulting chimeric species can have two or more sets of chromosomes derived from different parental species; this is called allopolyploidy (Stebbins, 1971; Song et al., 1995; Bennett, 2004; Hegarty and Hiscock, 2005). In contrast with allopolyploidy, which may occur in connection with hybridization between taxa that are not very closely related, hybrid speciation without a change in chromosome number may occur in cases where the parental species are closely related and their primary hybrid is somewhat fertile; this process is called homoploid hybrid stabilization (Rieseberg, 1997; Rieseberg et al., 2003; Abbott et al., 2005).

Polyploid evolution has been an important factor in the evolutionary history of land plants, and continues to be so also in extant lineages such as the plant genus Viola (Violaceae). In fact, the first report of an infrageneric series of polyploid levels was from Viola (Miyaji, 1913). The base chromosome number of Viola is believed to be x=6 or x=7, but the vast majority of north-temperate taxa have been shown to be paleo-allotetraploid with secondary base numbers of x=10 or x=12 (Nordal and Jonsell, 1998; Marcussen and Nordal, 1998; Karlsson et al., 2009). These are hereafter referred to as secondary diploids.

Further polyploidy based on these secondary diploid chromosome numbers has been demonstrated especially within the species-rich subsections of section Viola (Karlsson et al., 2009).

Within Section Viola subsection Rostratae Kupffer (sometimes treated as the separate section Trigonocarpea (Godr.) Vl. V. Nikitin), most species have retained the secondarily diploid chromosome number of 2n=20. However, subsequent polyploidization events have led to the formation of higher-ploids with chromosome numbers of 2n=40 (octoploid), 60 (dodecaploid) or even 58 (sub-dodecaploid); these are hereafter referred to as secondary tetraploids and (sub-)hexaploids, respectively. Nearly all of these secondary polyploids, a total of ten species, are native to western Eurasia, and their relatively recent polyploid parentages have been investigated in a series of cytological studies in the late 1950s and early 1960s (fig. 1) (Valentine 1950, 1958; Moore and Harvey 1961; Harvey, 1966).

The subsection consists of about fifty species with a northern temperate distribution in North America and Eurasia. Most species have white to dark lilac flowers and grow in woodlands. Subsection Rostratae is characterized primarily by primitive characters.

Phylogenetic analyses based on nuclear ribosomal Internal Transcribed Spacer (nrITS) sequences have recovered that the subsection is paraphyletic with respect to a number of other north-temperate groups (Ballard et al., 1999; Yoo et al., 2005). In Europe, where subsection Rostratae is morphologically most diverse, the subsection has traditionally been subdivided in a variable number of morphologically defined groups, usually at the series level. Series Rosulantes is characterized by having a basal rosette and flowers produced only from the lateral aerial shoots; this growth form is found also in other sections and may be considered as primitive within the genus. Series Mirabiles differs from the Rosulantes in producing flowers also from the basal leaf rosette, series Arosulatae in lacking the basal rosette altogether, and series Repentes in being stoloniferous and producing flowers from the rosettes. However, the recognition of series is problematic for

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15 two main reasons. First, the series typically define small groups of species by a very limited number of autapomorphies, thereby rendering the remaining groups paraphyletic and defined by synplesiomorphies only. Second, several of these series cannot be considered monophyletic because of the alloploid relationships between taxa of different series.

mirabilis

M reichenbachiana

A unknown sp.

B

elatior canina CC?

BC pumila

riviniana CD pseudo-mirabilis AB

MA

lactea BCE

Espeut

(1999) Clausen

(1927)

stagnina

C unknown sp.

D unknown sp.

E

series Mirabiles series Rosulantes series Arosulatae

2n = 20 2n = 40 2n = 58

Fig. 1. Hypotheses of relationships between different genome types (A, B, C, D, E and M) in species of Viola subsection Rostratae. Series affinity is indicated with shades of gray: series Mirabiles black; series Rosulantes dark grey; series Arosulatae light gray. Presumably extinct taxa are indicated with dashed lines. Data from Moore and Harvey (1961) except where indicated (Clausen, 1927; Espeut, 1999).

Series Arosulatae defines a small group of five western Eurasian species. Species of the series are specialists of temporarily flooded habitats, rather than woodland, and are easily characterized by lacking leaf rosettes and by their leaf and stipule characters.

Traditionally seven species have been recognized, but two (the East Asian V. acuminata and the submediterranean V. jordanii) must be omitted on the basis of having a leaf rosette and the quite different choices of habitat. The remaining five species are all Central European. Viola stagnina is the only secondary diploid with 2n=20, whereas three species are secondary tetraploids with 2n=40 (V. canina, V. elatior, V. pumila) next to a secondary sub-hexaploid with 2n=58 (V. lactea) (Moore and Harvey, 1961).

Especially the study by Ballard et al. (1999) indicated that the taxonomy of the genus Viola needs revision and that more molecular phylogenetic studies are called for. Although the nrITS region used by Ballard et al. (1999) was useful for recognizing infrageneric groups of the genus Viola, nrITS is generally not useful for examining evolutionary relationships among polyploid lineages. This is because recombination and concerted evolution between orthologous nrITS copies often lead to retention of only one copy type and erasion of the

A l l o p o l y p l o i d E v o l u t i o n a r y R e l a t i o n s h i p s o f E u r o p e a n R o s t r a t e V i o l e t s

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other parental copy (Wendel et al., 1995; Álvarez and Wendel, 2003). This is usually also the case in Viola, as nearly all investigated species have retained only one nrITS copy type regardless of ploidal level (Ballard et al., 1999; Malécot et al., 2007). The nrITS as a phylogenetic marker is therefore not suitable for recovering the reticulate relationships within the genus. Plastid markers generally also have the problem of retention of a single parental copy as these are usually uniparentally inherited in plants; furthermore, sequence variation in plastid markers is usually low (Corriveau and Coleman, 1988; Taberlet et al., 2007). Again, reticulate relationships therefore remain obscure.

Álvarez and Wendel (2003) suggested using single or low copy nuclear markers to circumvent the problem of concerted evolution causing misleading phylogenetic reconstructions of polyploid species. Phylogenetic analysis of paralogous and orthologous copies of single or low copy genes in alloploid species is also a good method to reveal the parental contributors to alloploid genomes. This method has been successfully applied in numerous studies (e.g. Popp and Oxelman, 2001; Smedmark et al., 2005).

We utilized the low copy nuclear Chalcone synthase (CHS) gene as a phylogenetic marker in Viola subsection Rostratae. As an independent dataset we chose the trnS-trnG intergenic spacer and intron as plastid phylogenetic marker, as this region proved to be sufficiently informative in Viola to assess interspecific relationships.

CHS is the first enzyme in the flavonoid synthesis pathway and is encoded by a small gene family (Durbin et al., 1995). Flavonoids are important secondary metabolites responsible for a multitude of tasks in plants, ranging from flower and fruit coloration and protection against UV radiation to pathogen defense and pollen development (Harborne, 1994). In Viola cornuta, three different CHS gene copies were found to be expressed from early stages of flower coloration onwards (Farzad et al., 2003). In general, genes of the CHS family consist of one intron flanked by two exons. There is high variation in the number of CHS copies among angiosperms. In asterids the number of CHS copies ranges from a single copy in Antirrhinum (Sommer and Saedler, 1986) to six copies in Ipomoea (Clegg and Durbin, 2003) and eight in Petunia (Koes et al., 1987). Similarly for the rosids, both Arabidopsis (Wang et al., 2007) and Populus (Tuskan et al., 2006) have two CHS copies, whereas both Vitis (Sparvoli et al., 1994; Jaillon et al., 2007) and Viola cornuta cultivars (Farzad et al., 2003; 2005) have three CHS copies.

We collected different CHS paralogues in species of Viola subsection Rostratae and analyzed these phylogenetically to 1) test earlier hypotheses about reticulate relationships of several allopolyploid taxa based on karyotype data in subsection Rostratae (e.g. between V. stagnina, a possible Dutch endemic, and its closest relatives), 2) make a comparison with a species phylogeny of Viola subsection Rostratae based on sequences of the plastid trnS-trnG intron and intergenic spacer to infer how many duplications of CHS took place during the evolution of Viola.

Materials and Methods

Taxon sampling

In total, 30 Viola taxa with a predominantly western European origin were sampled, of which 21 taxa belong to Viola subsection Rostratae. The nine taxa outside subsection Rostratae represent sections Andinium, Boreali-Americanae, Chamaemelanium, Erpetion,

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17 and Melanium and subsection Viola of section Viola. These species appeared to be either closely or more distantly related to the species of subsection Rostratae in a previous molecular phylogenetic study of Viola (Ballard et al., 1999). DNA was obtained from freshly collected material from the field and from herbarium collections.

For reconstruction of the CHS gene lineage tree, two different parts of the gene were sampled, the intron and exon 2. Exon 2 lineages available in Genbank from representatives of major Angiosperm clades were included in the analysis to find out whether the different CHS copies present in Viola are the products of recent or more ancient duplications. The following lineages were sampled: Gymnosperms: GbCHS (Ginkgo biloba, AY647263) and PsCHS (Pinus sylvestris, X60754); Monocots: IhCHS (Iris x hollandica, AB232914), HvCHS (Hordeum vulgare, X58339), and ZmCHS (Zea mays, AY728478, X60204); Core eudicots:

VvCHS (Vitis vinifera, AB015872, AB066275, EF192464, AM 454341, X75969); Rosids:

GmCHS (Glycine max, AY262686), PsCHS (Pisum sativum, D88263, D88262, D88261, D88260, X63333), PtCHS (Populus spp., DQ371804, EF147137, EF147091, DQ371802), VcCHS (V. cornuta cultivar, AY497407, AY497414); Asterids: AmCHS (Antirrhinum majus, X03710), DcCHS (Daucus carota, D16255), and PhCHS (Petunia hybrida, X14597).

The phylogenetic analyses performed were all rooted differently. There were several reasons for this. First of all, plastid sequences of non-Violaceae were not used for phylogenetic analyses. The Angiosperm Phylogeny Group topology was used to constrain the analyses instead (see below). For the plastid phylogeny, closely related genera of Viola were used as outgroups. Second, CHS intron sequences outside Viola could not be aligned with CHS intron Viola data because of too high sequence divergence. For the CHS intron analyses, we therefore tentatively used Viola CHS3 as outgroup. Third, CHS gene duplication events could only be assessed with a broad taxonomic sampling. For the CHS exon 2 analyses, we therefore used gymnosperm lineages for rooting.

DNA Extraction, Polymerase Chain Reaction Amplification, Cloning, and Sequencing Total genomic DNA was extracted using the Dneasy Plant Mini Kit (Qiagen, Hilden, Germany) and the cetyltrimethylammonium bromide (CTAB) method of Doyle JJ and Doyle JL (1987) with some modifications. Leaf material was ground using a Ratch Mill.

In total, 750 μl CTAB buffer (Doyle and Doyle, 1987) was added to the ground material together with proteinase K and RNase. After incubating for 30 minutes at 60^oC, 750 μl of chloroform-isoamyl alcohol (24:1) was added. The samples were briefly vortexed and then centrifuged for 10 minutes at 12,000 rpm. The upper aqueous layer was transported to a clean 2 ml tube. A total of 500 μl chloroform-isoamyl alcohol (24:1) was added and the samples were again centrifuged at 12,000 rpm. After 5 minutes spinning, the upper phase was transferred to a new 2 ml tube. The DNA was then precipitated by adding cold 500 μl isopropanol. The samples were shaken 5 to 10 minutes and subsequently centrifuged for 15 minutes at 12,000 rpm. The supernatant was removed and 70% ethanol was added.

The samples were subsequently shaken vigorously for 2 minutes, after which the ethanol was poured off. The remaining ethanol was removed by evaporation. The resulting DNA pellet was dissolved in 200 μl 0.1x Tris-EDTA buffer.

In total, one plastid region (trnS-trnG spacer and intron) and one nuclear region (CHS intron and exon 2) were amplified and sequenced. Polymerase chain reaction (PCR) amplification of the plastid spacer and intron was performed with primers designed by

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Shaw et al. (2005). For the trnS spacer, the primers trnS^GCU (5’-AGA TAG GGA TTC GAA CCC TCG GT-3’) and trnG2S (5’-TTT TAC CAC TAA ACT ATA CCC GC-3’) were used. The trnG intron was amplified with the primers trnG^UUC (5’-GTA GCG GGA ATC GAA CCC GCA TC-3’) and trnG2G (GCG GGT ATA GTT TAG TGG TAA AA).

The primers CHSX1F (5’-AGG AAA AAT TCA AGC GCA TG-3’) and CHSX2RN (5’- TTC AGT CAA GTG CAT GTA ACG -3’) designed by Strand et al. (1997) were used for amplifying the CHS intron. The primers CHS forward (5’-TAY CAR CAR GGN TGY TTY GC-3’) and CHS reverse (5’-GGR TGD GCD ATC CAR AAV A-3’) from Farzad et al. (2003) were used to amplify exon 2 of the CHS gene (fig. 2a). The generated intron and exon sequences did not overlap as the intermediate part turned out to be too large and too heterogeneous for this. PCR fragments for several Viola species of the CHS intron are shown in fig. 2b. Per individual, 12 clones were analyzed and consensus sequences were compiled from 3-7 individual clones.

Fig. 2. Amplified regions of chalcone synthase (CHS):

Fig 2a. Map of the two exons and interjected intron of the CHS gene. Primers and their binding sites are indicated for the CHS intron data set (black triangles) and the CHS exon 2 data set (white triangles).

exon 1 ~200 bp exon 2 ~1000 bp

CHSX1F CHSX2RN CHS forward CHS reverse

CHS gene:

CHS primer pairs:

CHS intron

PCR amplification conditions for the trnS spacer consisted of denaturing for 50 s at 95ôC, annealing for 1 min at 53ôC, and extension for 2 min at 72ôC. This cycle was repeated 35 times. The trnG intron was amplified with the same conditions except for the annealing temperature which was 56ôC. PCR amplification conditions for the CHS intron consisted of denaturing for 1 min at 95ôC, annealing for 90 s at 53ôC, and extension for 2 min at 72ôC. This cycle was repeated 35 times. The conditions for amplifying the CHS exon 2 consisted of denaturing for 45 s at 95ôC, annealing for 1 min at 55ôC and extension for 1 min at 72ôC. This cycle was repeated 40 times.

PCR products were purified using the Promega Wizard Purification System, cloned using the pGEM^®-T Easy Vector System and sequenced using the M13 primers with 30 s denaturing at 95ôC, annealing for 30 s at 50ôC, and extension for 1 min at 72 ôC. This cycle was repeated 35 times. PCR products were purified and analyzed on an ABI 377 (Applied

Fig. 2b. PCR products on 1%

agarose gel of different CHS intron copies found in Viola. Numbers correspond to the different CHS intron copies.

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19 Biosystems Inc., Foster City, CA) or a MegaBACE Sequence Analyzer 4.0 (Amersham Biosciences, Uppsala, Sweden) automated sequencer using the manufacturers’ protocols.

Phylogenetic analyses

DNA sequences were aligned using McClade 4.06 (Maddison DR and Maddison WP, 2003) with the pairwise alignment option and manual adjustment where necessary.

Individual insertion and deletion events were manually added as additional binary characters.

MrModeltest version 2.2 (Nylander, 2004) was used to find the best model of sequence evolution (Posada and Crandall, 1998). The models used for Bayesian analyses were the symmetrical model with separate gamma distributions and a separate proportion of invariant sites for CHS exon 2 (SYMIG model), the General Time Reversible model with gamma distribution for CHS intron (GTRG model), and the General Time Reversible model with gamma distribution and a separate proportion of invariant sites for trnS-trnG (GTRIG model). Maximum Parsimony (MP) analyses were carried out with PAUP* 4.0b10 (Swofford, 2003). Phylogenies were obtained using the heuristic search option, with twenty random sequence additions and Tree Bisection-Reconnection branch swapping.

After each sequence addition, a maximum of 10,000 trees was saved.

For MP, bootstrap support (Felsenstein, 1985) was calculated with 2,000 bootstrap replicates, using only ten random sequence additions each bootstrap replicate. After every random sequence addition replicate a maximum of 2,500 trees were saved. Bayesian inference analyses were performed using MrBayes 3.1 (Huelsenbeck and Ronquist, 2001).

Markov Chain Monte Carlo analyses (MCMC) were run for eight million generations with five simultaneous MCMCs, saving one tree per 100 generations. The burn-in values were identified using the program Tracer 1.3 (Rambaut and Drummond, 2004).

To convert the CHS gene tree composed of multiple paralogous lineages from allopolyploid taxa into a species tree to assess gene duplications, GeneTree version 1.3 (Page and Charleston, 1997) was used. The analyses were run with default settings.

GeneTree requires fully resolved organismal and gene trees as input. For the organismal tree, one of the 200,000 fully resolved most parsimonious trees (MPTs) of trnS-trnG data was chosen randomly. This analysis was constrained for all non violets to the latest angiosperm phylogeny topology as depicted on the Angiosperm Phylogeny Website (version 8, June 2007) (www.mobot.org/MOBOT/research/Apweb/). For the CHS exon 2 gene tree, the 95% most probable Bayesian tree was used.

Homology Assessment of CHS Copies

The CHS lineages found were assigned to different copies based on size, sequence divergence and phylogenetic position (Helariutta et al., 1996; Doyle and Davies, 1998;

Smedmark et al., 2005). CHS fragments within one species with only minor divergence and gaps were interpreted as alleles. The different CHS copies of V. cornuta published by Farzad et al. (2003) always ended up in a single clade in all analyses performed here. We therefore used a single representative sequence only. When size difference and sequence divergence were more apparent, e.g. by the presence of large indel events, the CHS fragment was treated as a paralogous copy. Our classification of alleles and paralogous copies was further confirmed by topological positions in the phylogenies obtained.

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Results

trnS-trnG

MP analyses of the trnS-trnG alignment produced a total of 200,000 MPTs with 537 steps (consistency index [CI] = 0.8239; retention index [RI] = 0.7312). The majority rule consensus tree (data not shown) has a similar topology as the Bayesian tree (fig. 3). We plotted both the Bootstrap Support values (BS) and Posterior Probability Index values (PPI) on the latter. All species sampled of Viola subsection Rostratae ended up in five different, poorly to well supported subclades (<50–98% BS; 0.56–1.00 PPI). The largest subclade consists of V. stagnina, V. elatior, V. lactea, V. canina, V. sieheana, V. jordanii, V. oligyrtia, V.

rupestris, and V. pumila.

V. cornuta

V. pedatifida V. sororia V. pubescens V. banksii

V. suavis

V. acuminata

V. uliginosa V. pseudo-mirabilis V. odorata

V. caspia V. mirabilis V. willkommii

V. riviniana f. riviniana V. riviniana f. purpurea V. reichenbachiana V. pumila V. grayii V. ovato-oblonga

V. jordanii V. oligyrtia V. rupestris

V. canina V. sieheana

V. stagnina var. lac.

V. stagnina var. stag.

V. lactea

V. elatior

subsect. Boreali-Americanae

subsect. Rostratae subsect. Viola section Chamaemelanium

section Chamaemelanium section Erpetion

0.66 / <50 0.66 / <50 0.67 / <50 0.99 / 55

1.00 / 92 1.00 / 85

0.55 / <50 1.00 / 98

V. biflora

V. alba

section Melanium

section Viola 0.93 / <50

0.56 / <50 0.93 / 77

0.88 / 54

V. reichei Cubelium concolor Allexis batangae

1.00 / 100

0.80 / 72 0.99 / 72

Outgroups (Violaceae) section Chilenium V. rosulata section Andinium 1.00 / 100

V. dasyphylla

Fig. 3. Plastid trnS-trnG phylogeny of Viola.

(MP majority rule consensus of 200,000 trees; ci = 0.8239, ri = 0.7312, 537 steps. Numbers on branches refer to PPI and BS values).

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21

Fig. 4. CHS intron gene lineage tree.

(MP majority rule consensus of 200,000 trees; ci = 0.7828 ri = 0.8824, 921 steps. Numbers on branches refer to PPI and BS values) The genomes described in Figure 1 which could be recognized are indicated. Taxa in bold refer to extant secondary diploids.

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Ginkgo

Vitis Antirrhinum Daucus Petunia Pisum Glycine Populus V. reichenbachina V. uliginosa V. cornuta V. grayii V. stagnina V. canina V. willkommii V. pseudo-mirabilis V. mirabilis V. biflora V. pubescens

V. riviniana f. riviniana V. riviniana f. purpurea V. reichenbachiana V. uliginosa V. cornuta V. grayii V. stagnina V. canina V. willkommii V. pseudo-mirabilis V. mirabilis V. biflora V. pubescens

V. riviniana f. riviniana V. riviniana f. purpurea

V. grayii V. stagnina V. canina V. willkommii V. pseudo-mirabilis V. mirabilis V. rivinianaf. riviniana V. rivinianaf. purpurea V. biflora V. pubescens V. cornuta V. grayii IrisZea Hordeum Vitis Antirrhinum Daucus Petunia Pisum Glycine

V. grayii V. stagnina V. canina V. willkommii V. pseudo-mirabilis V. mirabilis V. riviniana f. riviniana V. riviniana f. purpurea ZeaIris

Hordeum

V. grayii V. stagnina V. canina V. willkommii V. pseudo-mirabilis V. mirabilis V. riviniana f. riviniana V. riviniana f. purpurea V. uliginosa V. cornuta V. grayii V. stagnina V. canina V. willkommii V. pseudo-mirabilis V. mirabilis V. riviniana f. riviniana V. riviniana f. purpurea V. uliginosa V. cornuta V. grayii V. stagnina V. canina V. willkommii V. pseudo-mirabilis V. mirabilis V. riviniana f. riviniana V. riviniana f. purpurea V. uliginosa V. cornuta V. biflora V. pubescens V. reichenbachianaPopulus

Pinus

Gene duplication event Gene loss or undiscovered lineage Extant genelineage

Clade 2 Clade 1

Fig. 5. Reconciled tree of CHS exon 2 gene lineage and trnS-trnG phylogenies (constrained with APG topology) showing duplication/

loss events, reconstructed with GeneTree version 1.3 (Page and Charleston, 1997).

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23 CHS intron and exon 2

Three copies of the CHS intron were found in the sampled species of Viola subsection Rostratae (figs. 2b and 4). Copies 1 (CHS1, 600 base-pairs [bp]) and 2 (CHS2, 735–1100 bp) have a relatively similar sequence identity and were found in all Viola species sampled with the exception of V. biflora, V. banksii, and V. pubescens. The third copy was not found in all species and was the largest sized (CHS3, 775–1,160 bp). The CHS3 copy is probably present in more taxa, but amplification failures probably led to an under sampling of this particular copy. It differed quite substantially from the other two copies in size and sequence similarity. In contrast with CHS1, multiple paralogs/orthologs were found in CHS2 and CHS3.

The complete CHS intron alignment consisted of 2,980 bp after exclusion of a 64 bp segment that was too variable for proper alignment. A total of 302 characters were phylogenetically informative, of which 12 were indel characters (indels varying in size between 5 and 422 bp). Two indels, found in CHS1 and CHS2, seemed to be the result of slip strand mispairing as many repeats were found in these regions. The first (TGATTT) and second repeat (TGTT) were repeated up to four times. The other ten indels lacked a repetitive structure. Most of the indels occurred in CHS2. In CHS3, two large indels were found of 182 and 422 bp, respectively.

MP analyzed of CHS intron sequences produced 200,000 MPTs (921 steps, CI = 0.7828, RI = 0.8824). The majority rule consensus tree (fig. 4) had a similar topology as the Bayesian tree (data not shown).

At least two different copies of CHS exon 2 were found in Viola. Both copies had a similar size (984 bp) and were retrieved from almost all Viola species analyzed.

They differed substantially in sequence similarity. Of the 984 bp retrieved, 498 were phylogenetically informative. One autapomorphic gap was found. Bayesian analyses of CHS exon 2 sequences produced a topology similar to MP (data not shown), in which two main clades were present comprising the different copies of CHS exon 2.

Reconciliation of Gene Tree and Species Tree

A reconciled tree (fig. 5) reconstructed with the program GeneTree (Page and Charleston 1997) was used to visualize CHS exon 2 duplications during the evolution of Viola. It seems that assuming six CHS gene duplication events is sufficient to make the gene and species tree congruent.

Discussion

Polyploidy in Viola Subsection Rostratae

The internal topology of the CHS2 intron clades (fig. 4) is in general agreement with previously inferred relationships between parental species and their allopolyploid hybrids in Viola subsection Rostratae (Moore and Harvey, 1961). Moore and Harvey (1961) could recognize the parental karyotypes in the genomes of artificially constructed allopolyploid Viola hybrids by the unique size and shape of the chromosomes. Subsequently, they

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used observations on chromosome pairing to formulate hypotheses regarding the origin of allopolyploids (fig. 1). In their study, five different types of genomes (A–E) could be recognized, each referring to the secondary diploid level (2n=20). Only the A and C genome occurred in extant secondary diploids, namely in V. reichenbachiana (A) and V. stagnina (C). The other four genome types were found only in combination with other genome types in the secondary tetraploid (2n=40) or sub-hexaploid taxa (2n=58).

From this, they concluded that V. stagnina (C) contributed a C genome to V. canina (BC) and its close relative V. lactea (BCE) and possibly also to V. pumila (CD). Similarly, V.

reichenbachiana (A) would have contributed an A genome to V. riviniana f. riviniana (AB).

Thus, the species possessing the B genome was involved in the origin of both V. riviniana f. riviniana (AB) and V. canina (BC). The authors attributed the three “missing” genomes (B, D, and E) to secondary diploids species that might have become subsequently extinct, at least in Europe. Viola elatior was not included in this study.

In the CHS2 intron tree, V. canina was found to have one orthologue in common with V. stagnina (corresponding to genome C) and a second in common with V. riviniana (corresponding to genome B). The second orthologue of V. riviniana was found to be closely related to V. reichenbachiana (corresponding to genome A). Like V. canina, V. pumila had one orthologue in common with V. stagnina (genome C) while its second copy was found to be closely related to V. canina and V. riviniana suggesting that genome D could have been derived from genome B.

The phylogenetic position of the gene lineages retrieved from V. elatior suggests that this particular species probably contains the C genome because its CHS2 intron copy ended up close to V. stagnina. The chromosome number of V. elatior (2n=40) suggests that it is a secondary tetraploid, but we were not successful in detecting more than one orthologue in this species. Unpublished isozyme studies also reveal a lower number of allozymic bands than usual in species of this ploidal level. These findings, together with earlier observations of quadrivalents in the meiosis of the species (Clausen, 1927) indicate that V. elatior may be an autopolyploid derivative of some stagnina-like ancestral species possessing the C genome.

In contrast with the Arosulatae series, which was found to be monophyletic for CHS1 and part of CHS2, CHS lineages retrieved from species assigned to the Rosulantes and Mirabiles series did hardly ever end up in the same clades. This is probably caused by the fact that the morphological characters used to delimit these series are phylogenetically uninformative. Unknown hybridization and polyploidisation events within and between these series probably also cause paraphyly.

The small series Mirabiles consists of one secondarily diploid (2n=20) species, V.

mirabilis, and two secondary tetraploids (2n=40). The local endemic V. pseudo-mirabilis of Les Grands Causses in southern France has been variously interpreted on morphological grounds as an intermediate between V. mirabilis and V. riviniana (Valentine et al., 1968) or as a polyploid derivative of V. mirabilis and V. reichenbachiana (Espeut, 1999). The latter view has later been confirmed by own unpublished isozyme data and a chromosome count of 2n=40 (Verlaque and Espeut, 2007). In the present study, V. pseudo-mirabilis ends up as sister to V. riviniana (61% BS; 0.99 PPI), which is not in contradiction to the previous findings because V. riviniana is itself a polyploid derivative of V. reichenbachiana.

The other secondary tetraploid Mirabiles species, V. willkommii, endemic to northern Spain, is morphologically rather similar to V. mirabilis but differs considerably in choice of habitat. This particular species is a secondary tetraploid and has been supposed to have

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25 originated from V. mirabilis and V. rupestris (Marcussen, personal communication). Our CHS data confirm the parentage of V. mirabilis but the second parent of V. willkommii remains unclear. Two paralogues of the CHS2 intron were retrieved from V. willkommii, of which one ended up in a strongly supported clade (96% BP; 0.97 PPI) with V. mirabilis and the other in an unresolved polytomy. The CHS1 intron fragment retrieved from V.

willkommii ended up in a basal dichotomy with the lineage of V. rupestris. Future genome type data should be collected of the Mirabiles series to confirm hypotheses about reticulate relationships suggested by the CHS gene lineages obtained here.

Closest Relatives of Viola stagnina

Viola subsection Rostratae as mentioned before has been taxonomically subdivided in series Arosulatae, Mirabiles, Repentes and Rosulantes (Valentine, 1958). Viola stagnina is considered to be a member of the Arosulatae series together with V. canina, V. elatior, V.

lactea, and V. pumila, based on the lack of a basal leaf rosette. This taxonomic placement is supported by the fact that in our study, two Arosulatae clades are present in the CHS intron gene lineage tree (fig. 4), one consisting of CHS1 intron lineages and one of CHS2 intron lineages. These lineages were all retrieved from species assigned to the Arosulatae series.

Based on previous morphological and karyological studies and our results, we can conclude that the closest relatives of V. stagnina are V. pumila, V. elatior, V. canina and V.

lactea. Fingerprinting techniques are currently being applied to assess gene flow between different European populations of V. stagnina and its close relatives to determine whether a Dutch variety of V. stagnina deserves a different taxonomic status.

CHS Lineage Diversification in Viola

In the reconciled tree (fig. 5), two main clades are present. Unfortunately, GeneTree does not provide statistical support for individual nodes. The congruence in topology with Huang et al. (2004) and Yamazaki et al. (2001) indicates that a general phylogenetic signal was recovered, though. The first clade consists of monocot, core eudicots and Viola representatives. This clade indicates that at least one duplication event in the CHS gene family took place before the split between the monocots and the eudicots. The second main clade consists of rosid, asterid and Viola representatives. This indicates that another duplication event in the CHS gene family took place during the split between the core eudicots and rosids/asterids. Similar results were also found by Huang et al. (2004).

Yang and Gu (2006) also describe multiple rounds of CHS gene duplications during the evolution of the angiosperms. According to these authors, the most ancestral gene lineages originated during the divergence of different plant families, such as Solanaceae, Convolvulaceae and Asteraceae in a first round of duplications. Derived CHS genes further duplicated and diverged, which led to the occurrence of various CHS plant family specific genes in subsequent rounds of duplication.

The CHS intron gene lineage tree suggests that three CHS copies are present in Viola, whereas the CHS exon 2 gene lineage tree (data not shown) only contains two copies. The oldest duplication event in clade I (fig. 5) suggests the possibility of the presence of a third copy of CHS in Viola. This might also explain why CHS intron copies 1 and 2 have a close resemblance. The fact that not all copies were retrieved does not mean they are not there

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but could explain why taxa from different sections end up nested in section Viola in the reconciled tree. Our interpretation of the CHS paralogues in Viola is different from Farzad et al. (2003, 2005). We consider the CHS paralogues in V. cornuta to be different alleles whereas the latter study identified them as different copies. The plant analyzed by Farzad et al. (2003, 2005) was a garden cultivar of hybrid origin. The nominal species is in itself a high-polyploid (Marcussen et al., forthcoming). Furthermore, our analyses of a larger sample of different Viola lineages showed that the interpretation of Farzad et al. (2005) was incomplete. Farzad et al. (2005) showed that the three CHS paralogous in V. cornuta are all still expressed and fully functional. Expression patterns were found to be slightly different, which might indicate subfunctionalization. Subfunctionalization of duplicate CHS genes in angiosperms appears to have happened by differentiation of their regulatory elements (Yang and Gu, 2006). It would be interesting to further investigate the mechanisms of subfunctionalization in a wider array of Viola species.

Acknowledgements

We would like to acknowledge T. Marcussen for providing material and for his valuable feedback on earlier versions of this manuscript. Furthermore, we like to acknowledge P. van Beers, R.L. Eckstein, H.

den Held, F. Hellberg, W.J. Holverda, the late R. van der Meijden, R. van Moorsel, I. Nordal, P.B. Pelser, N. Schidlo, E.J. Weeda, and B. Wijlens for providing material and assistance in the field; Kew Botanical Gardens for providing DNA samples; and M.C.M. Eurlings, B.J. van Heuven, L. McIvor, S. Bollendorff, and D.L.V. Co for technical support. We would like to acknowledge the following organizations and departments for providing access to plant material and collecting permits: Staatsbosbeheer, Natuurmonumenten, Overijssels Landschap, and Kalmar county environmental department.

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27

Chapter

3

Viola montana and V. persicifolia (Violaceae): two names to be rejected

²

J. Danihelka, K. van den Hof, T. Marcussen and B. Jonsell

T

he taxonomic and nomenclatural histories of Viola elatior Fries (1828), V. pumila Chaix (1785) and V. stagnina Kit. ex Schult.

(1814) in central and western Europe are discussed. The names V.

stagnina and V. elatior are lectotypified with specimens corresponding to the current use of these names. The neglected lectotypification of V.montana L. (1753) from 1988 with a specimen referable to V.

elatior is briefly reviewed. The name V. persicifolia Schreb. (1771), used in some floras instead of V. stagnina, is analyzed in detail, and we conclude that it should be interpreted as referring to V. elatior as well. The use of V. persicifolia and V. montana, representing the correct name for the species widely known as V. elatior, has been notoriously confused for two centuries, and we herein recommend to reject these two names in order to assure nomenclatural clarity and stability.

Keywords: Europe, nomenclature, typification, Viola elatior, V.

stagnina, Viola subsect. Rostratae

2Danihelka et al., submitted to Taxon (in review).

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Introduction

Viola subsect. Rostratae Kupffer (= V. sect. Trigonocarpea Godron) is represented in Europe by five arosulate species, often referred to as V. ser. Arosulatae Borbás (van den Hof et al., 2008). Viola canina L. (2n = 40), the commonest one, has a wide distribution range reaching from the Iberian Peninsula in the west to Lake Baikal in the east. It is extremely morphologically variable, and its intraspecific classification is still in dispute.

Viola lactea Sm. (2n = 58), in contrast, is a strongly oceanic species confined to the British Isles, the northern parts of the Iberian Peninsula, western France, and Belgium. The three remaining species, in recent literature known as V. elatior Fries (2n = 40), V. pumila Chaix (2n = 40), and V. stagnina Kit. ex Schult. (or V. persicifolia Schreb.; 2n = 20), have wide distribution ranges reaching from the British Isles and eastern France eastwards to western or central Siberia. In central Europe they are often confined to the floodplains of the large lowland rivers. The taxonomy and ecology of the three floodplain violets in Central Europe was recently reviewed by Eckstein et al. (2006a). In the course of our studies, we have encountered nomenclatural difficulties that will be dealt with herein.

Viola montana

Herbarium specimens of V. elatior collected in the late 18th and early 19th centuries have been frequently identified as V. montana L. (Sp. Pl. 2: 935. 1753), which is in conflict with the prevailing current use of this Linnean name for certain morphotypes of V. canina.

These different interpretations can be traced back to a redefinition of V. montana in the second edition of Flora suecica (Linnaeus, 1755) and subsequently in the second edition of Species Plantarum. The use of the name V. montana has been repeatedly discussed.

Some authors have suggested that the name V. montana originally referred mainly to the plant currently known as V. elatior (e.g. Fries, 1828; Neilreich, 1859; Borbás, 1892;

Wilmott, 1916; Lindberg, 1958). Nikitin (1988) reviewed the nomenclatural history of V.

montana and proposed a lectotype (Herb. Linn. No. 1052.13, LINN) referable to V. elatior.

This lectotypification is in accordance with the protologue and should not be overruled.

However, only a few authors apart from Nikitin seem to have accepted its consequences (e.g., Chen Zousheng et al., 2007) and replaced V. elatior by V. montana, while many other national checklists and floras published after 1988 preferred nomenclatural stability and clarity to correctness, and continued using V. elatior. The replacement of a well established name by another name that was only rarely used in its original sense after the 1820’s is undesirable and would destabilize nomenclature. Therefore we have decided to propose V. montana for rejection, as already announced by Kirschner and Skalický (1989).

Viola montana L., Sp. Pl. 2: 935. 1753, nom. utique rej. prop. (van den Hof et al., Taxon:

in review³).

Ind. loc.: “Habitat in Alpibus Lapponiae, Austriae, Baldo.”

Lectotypus (vide Nikitin in Bot. Žurn. 73: 1541. 1988): “Viola 10 / montana” (Herb.

Linn.

No. 1052.13, LINN, vide http://www.linnean-online.org/11110/).

3Chapter 4 of this thesis.